A soluble cell-free system which catalyzes semi-conservative SV40 DNA replication of exogenously added SV40 form I DNA has been developed from African green monkey kidney or CV-1 cells infected with this virus. The DNA synthesis required Mg ions, ATP, all four dNTP, and exogenous SV40 form I DNA as a template and yielded SV40 form I and form II DNA as products. The reaction was inhibited by N-ethylmaleimide and salt, but not by ddTTP. These results suggest involvement of DNA polymerase alpha in SV40 synthesis in vitro (as in vivo). This SV40 DNA-dependent SV40 replication also seems to depend on the product of a gene of SV40, i.e., SV40 large T-antigen. These findings provide for an in vitro complementation assay for SV40 large T-antigen.